Project Summary Carbapenem-resistant Enterobacteriaceae (CRE), in particular Klebsiella pneumoniae carbapenemase (KPC)- producing strains, have emerged worldwide as major pathogens, causing diverse infections that are resistant to most antibiotic classes. Ceftazidime-avibactam (CAZ-AVI) is a new ?-lactam/?-lactamase inhibitor combination agent that is active against KPC-producing Enterobacteriaceae (KPC-E) in vitro. Data are still emerging for CAZ-AVI as treatment against KPC-E infections, but the agent is endorsed as a frontline option due to potent activity in vitro, favorable safety profile, and the shortage of alternative regimens. Through April 2017, we treated 77 CRE-infected patients with CAZ-AVI, 53 of whom were infected with sequence type (ST)-258 KPC-K. pneumoniae (KPC-Kp). CAZ-AVI resistance emerged in ST-258 KPC-Kp isolates from 8 patients. CAZ-AVI resistant isolates carried mutant blaKPC- 3 genes that encoded variant KPC enzymes. The most common KPC-3 variant had a tyrosine for aspartic acid substitution within the ? loop at Ambler position 179 (D179Y). In some isolates, D179Y was combined with a methionine for threonine substitution at position 243 (T243M). D179Y and D179Y/T243M directly conferred CAZ- AVI resistance, as proven by deletion of mutant genes in Kp clinical isolates and transfer of mutant genes into susceptible E. coli. blaKPC-3 mutations also significantly reduced meropenem (MEM) and aztrenoam (AZT) minimum inhibitory concentrations (MICs). In time-kill studies in vitro, we demonstrated that CAZ-AVI+MEM exerted synergy against KPC-Kp clinical isolates, and suppressed CAZ-AVI resistance. Our primary objectives in this project are to understand biochemical and structural mechanisms by which clinically relevant KPC variants mediate susceptibility or resistance to CAZ-AVI, MEM and AZT, and mechanisms of synergy between CAZ-AVI and the other agents. In specific aim 1, we will measure expression of bla encoding KPC-2, KPC-2/D179Y, KPC-3, KPC- 3/D179Y, and KPC-3/D179Y/T243M, and the respective proteins (aim 1a). Next, we will use purified proteins to assess steady state and pre-steady state enzyme kinetics with AVI, CAZ, MEM and AZT, and characterize reaction intermediates by timed electrospray ionization mass spectrometry (aim 1b). Then, we will generate molecular models and determine crystal structures of apo and complexed KPC-3, KPC-3/D179Y and KPC- 3/D179Y/T243M with CAZ and MEM (aim 1c). In aim 2, we will determine if CAZ-AVI+MEM and CAZ-AVI+AZT exert synergy and suppress CAZ-AVI resistance in vitro (aim 2a), and verify that synergy occurs in a mouse thigh model of KPC-E infection (aim 2b). Finally, we will measure binding of CAZ-AVI, MEM and AZT, administered alone and in combination, to penicillin binding proteins (aim 2c). Taken togerther, results will provide unique insights into mechanisms of action for naturally evolved KPC variants, biochemical and structural interactions between KPCs and antimicrobial agents, and mechanisms of synergy between CAZ-AVI and other ?-lactams. Data will be useful for developing strategies to optimize the clinical utility of CAZ-AVI, carbapenems and AZT, and in designing more effective ?-lactams and ?-lactamase inhibitors in the future.